Glycosphingolipid Characterization and Antibody Binding Assay

The reference glycosphingolipids were isolated and characterized by mass spectrometry and proton NMR as described (15 (link)). Thin layer chromatography was done on aluminum- or glass-backed silica gel 60 high performance thin layer chromatography plates (Merck). Glycosphingolipid mixtures (40 μg)or pure compounds (2–8 μg) were eluted using chloroform/methanol/water (60:35:8, v/v/v) as a solvent system. Glycosphingolipids were detected by the anisaldehyde reagent (15 (link)) or the resorcinol reagent (16 (link)).
The mouse monoclonal antibodies tested for binding to the acid glycosphingolipids of hESC in the chromatogram binding assay are given in supplemental Table S2. Binding of antibodies to glycosphingolipids separated on thin layer chromatograms was performed as described by Barone et al. (10 (link)). In short, glycosphingolipids were separated on aluminum-backed thin layer plates, and after drying the chromatograms were dipped for 1 min in diethylether/n-hexane (1:5, v/v) containing 0.5% (w/v) polyisobutylmethacrylate (Sigma-Aldrich) for 1 min. Thereafter, the chromatograms were soaked in PBS, pH 7.3, containing 2% bovine serum albumin and 0.1% NaN₃ (solution A) for 2 h at room temperature. Suspensions of monoclonal antibodies (the dilutions used for each antibody are given in supplemental Table S2) were gently sprinkled over the chromatograms, followed by incubation for 2 h at room temperature. After washing with PBS followed a second 2-h incubation with ¹²⁵I-labeled rabbit anti-mouse antibodies (DakoCytomation Norden A/S, Glostrup, Denmark) (labeled by the Iodogen method according to the manufacturer's (Pierce) instructions), diluted to 2 × 10⁶ cpm/ml in solution A. Finally, the plates were washed six times with PBS. Dried chromatograms were autoradiographed for 12–24 h using XAR-5 x-ray films (Eastman Kodak).